Human respiratory syncytial virus (RSV) is an important agent of pediatric respiratory tract disease of worldwide prevalence which is responsible for considerable morbidity and mortality, and which currently lacks an available vaccine. Its poor growth in tissue culture and most experimental animals and the instability of the virus particle have been major impediments to elucidating the molecular biology and pathogenesis of RSV and to developing vaccines. We recently developed the capability to produce infectious respiratory syncytial virus (RSV) by the intracellular coexpression of cDNAs encoding a complete RSV replicative intermediate RNA (antigenome) and the four RSV proteins that we previously found were necessary and sufficient to produce nucleocapsids competent for transcription and RNA replication, namely the N, P, L and M2 ORF1 proteins. This completed the critical goal of developing a method for the direct engineering of this important, uncontrolled pathogen. Our present goal is to use this system to understand RSV molecular biology and pathogenesis, and to identify novel types of mutations of interest for inclusion in a live recombinant vaccine virus (the construction of live attenuated recombinant vaccine viruses is also described in an accompanying report by Murphy et al). The first step is to investigate whether some of various individual RSV genes can be knocked out, and to determine what effects ensue. Translational stop codons were introduced into the translational open reading frame (ORF) of the NS1 or NS2 gene. The former could not be recovered into virus, whereas the latter was recovered as a recombinant which produced pin point plaques and grew more slowly in tissue culture. The SH gene also was knocked out, in this case by deleting the entire gene. This produced a virus which formed plaques that were larger than wild type. The SH-minus virus grew as well or better than wild type in tissue culture, whereas in mice it grew as well as wild type in the lower respiratory tract but was significantly restricted in the upper respiratory tract. This latter phenotype would be highly desirable in vaccine virus, since restricted replication of a vaccine virus in the upper respiratory tract would provide greater safety for the very young infant. A number of other modifications hold promise for improving a vaccine virus and remain to be tested. These experiments also will provide considerable insight into the basic mechanisms of viral gene expression and replication.